**Application of CRISPR**/**Cas9-Based Reverse Genetics in** *Leishmania braziliensis***: Conserved Roles for HSP100 and HSP23**

**Vanessa Adaui 1,2,3,**† **, Constanze Kröber-Boncardo 1,**† **, Christine Brinker <sup>1</sup> , Henner Zirpel 1,4 , Julie Sellau <sup>1</sup> , Jorge Arévalo <sup>2</sup> , Jean-Claude Dujardin 3,4,5,6 and Joachim Clos 1,\***


Received: 4 September 2020; Accepted: 25 September 2020; Published: 30 September 2020 -

**Abstract:** The protozoan parasite *Leishmania* (*Viannia*) *braziliensis (L. braziliensis*) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of *L. braziliensis* through reverse genetics analyses has so far lagged behind in comparison with Old World *Leishmania* spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in *L. braziliensis* that was previously developed for Old World *Leishmania major* and New World *L. mexicana* species. As proof of principle, we demonstrate the targeted replacement of a transgene (*eGFP*) and two *L. braziliensis* single-copy genes (*HSP23* and *HSP100*). We obtained homozygous Cas9-free *HSP23* and *HSP100*-null mutants in *L. braziliensis* that matched the phenotypes reported previously for the respective *L. donovani* null mutants. The function of *HSP23* is indeed conserved throughout the Trypanosomatida as *L. major HSP23* null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of *L. braziliensis* by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite's biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.

**Keywords:** *Leishmania braziliensis*; reverse genetics; CRISPR–Cas9; gene targeting; phenotyping; heat shock proteins
